Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS) production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-)/hydrogen peroxide (H2O2) release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 µM). This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM) restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

Selective activation of PI3Kalpha/Akt/GSK-3beta signalling and cardiac compensatory hypertrophy during recovery from heart failure.

AIMS: Activation of phosphoinositide-3 kinase (PI3K) is essential for cell growth, relating to adaptive and maladaptive cardiac hypertrophy. This longitudinal canine study was designed to investigate the role of PI3Kalpha and PI3Kgamma in cardiac remodelling during congestive heart failure (CHF) and cardiac recovery (CR). METHODS AND RESULTS: All dogs were surgically instrumented. Congestive heart failure was induced by cardiac pacing for 3-4 weeks and CR was allowed by terminating pacing for 5-6 weeks after induction of HF. Control dogs had sham surgery, but did not undergo pacing. Left ventricular (LV) contractile function was depressed in CHF and restored to 80-90% of the normal level in CR, with a 25% increase in LV weight. The expression of PI3Kgamma was increased four-fold in CHF, but returned to control levels in CR. In contrast, the expression of PI3Kalpha in CHF was not different from that in controls, but increased three-fold in CR and was accompanied by increases in phosphorylation of Akt (five-fold), GSK-3beta (five-fold), beta-catenin (three-fold), mTOR (two-fold), and P70S6K (two-fold). CONCLUSION: Our results indicate that PI3K isoforms are regulated differently during the course of CHF/CR and that the selective activation of PI3Kalpha, through Akt, GSK-3beta, and mTOR signalling pathways, may be involved in the development of cardiac compensatory hypertrophy and functional restoration.

Restoration of impaired endothelium-dependent coronary vasodilation in failing heart: role of eNOS phosphorylation and CGMP/cGK-I signaling.

In congestive heart failure (CHF), coronary vascular relaxation is associated with endothelial dysfunction and nitric oxide (NO) deficiency. This study explored the reversibility of this process in hearts recovering from CHF and its related mechanisms. Dogs were chronically instrumented to measure cardiac function and coronary blood flow (CBF). Heart failure was induced by right ventricular pacing at 240 beats/min for 3-4 wk, and cardiac recovery (CR) was allowed by the termination of cardiac pacing for 3-4 wk after the development of CHF, in which left ventricular contractile function was restored by 80-90%. The endothelium-dependent CBF response to bradykinin and acetylcholine was depressed in CHF and fully restored in CR. Myocardial NOx (nitrate/nitrite), endothelial NO synthase (eNOS) mRNA expression, total protein, and phosphorylated eNOS decreased significantly in failing hearts. However, myocardial NOx recovered to 78% of control and phosphorylated eNOS was fully restored in CR, despite the fact that eNOS mRNA expression and protein levels remained lower than control. Furthermore, the endothelium-independent CBF response to nitroglycerin did not change in CHF; however, it increased by 75% in CR, in conjunction with a near threefold increase in the phosphorylation of vasodilation-stimulated phosphoprotein (VASP) at Ser(239) in recovering hearts. Thus the complete restoration of endothelium-dependent coronary vascular relaxation during cardiac recovery from CHF was mediated by 1) a restoration of phosphorylated eNOS for partial recovery of the NO production and 2) an increase in cGMP/cGMP-dependent protein kinase-I pathway signaling activity for the enhancement of coronary vascular smooth muscle relaxation in response to NO.

Cardiac diastolic dysfunction in conscious dogs with heart failure induced by chronic coronary microembolization.

Left ventricular (LV) diastolic dysfunction is a fundamental impairment in congestive heart failure (CHF). This study examined LV diastolic function in the canine model of CHF induced by chronic coronary embolization (CCE). Dogs were implanted with coronary catheters (both left anterior descending and circumflex arteries) for CCE and instrumented for measurement of LV pressure and dimension. Heart failure was elicited by daily intracoronary injections of microspheres (1.2 million, 90- to 120-microm diameter) for 24 +/- 4 days, resulting in significant depression of cardiac systolic function. After CCE, LV maximum negative change of pressure with time (dP/dt(min)) decreased by 25 +/- 2% (P < 0.05) and LV isovolumic relaxation constant and duration increased by 19 +/- 5% and 25 +/- 6%, respectively (both P < 0.05), indicating an impairment of LV active relaxation, which was cardiac preload independent. LV passive viscoelastic properties were evaluated from the LV end-diastolic pressure (EDP)-volume (EDV) relationship (EDP = be(alpha*EDV)) during brief inferior vena caval occlusion and acute volume loading, while the chamber stiffness coefficient (alpha) increased by 62 +/- 10% (P < 0.05) and the stiffness constant (k) increased by 66 +/- 13% after CCE. The regional myocardial diastolic stiffness in LV anterior and posterior walls was increased by 70 +/- 25% and 63 +/- 24% (both P < 0.05), respectively, after CCE, associated with marked fibrosis, increase in collagen I and III, and enhancement of plasminogen activator inhibitor-1 (PAI-1) protein expression. Thus along with depressed LV systolic function there is significant impairment of LV diastolic relaxation and increase in chamber stiffness, with development of myocardial fibrosis and activation of PAI-1, in the canine model of CHF induced by CCE.

Exhaustion of the Frank-Starling mechanism in conscious dogs with heart failure induced by chronic coronary microembolization.

The role of the Frank-Starling mechanism in the regulation of cardiac systolic function in the ischemic failing heart was examined in conscious dogs. Left ventricular (LV) dimension, pressure and systolic function were assessed using surgically implanted instrumentations and non-invasive echocardiogram. Heart failure was induced by daily intra-coronary injections of microspheres for 3-4 weeks via implanted coronary catheters. Chronic coronary embolization resulted in a progressive dilation of the left ventricle (12+/-3%), increase in LV end-diastolic pressure (118+/-19%), depression of LV dP/dt(max) (-19+/-4%), fractional shortening (-36+/-7%), and cardiac work (-60+/-9%), and development of heart failure, while the LV contractile response to dobutamine was depressed. A brief inferior vena caval occlusion in dogs with heart failure decreased LV preload to match the levels attained in their control state and caused a further reduction of LV dP/dt(max), fractional shortening, stroke work and cardiac work. Moreover, in response to acute volume loading, the change in the LV end-diastolic dimension-pressure (DeltaLVEDD-DeltaLVEDP) curve in the failing heart became steeper and shifted significantly to the left, while the increases in LV stroke work and cardiac work were blunted. Thus, our results suggest that the Frank-Starling mechanism is exhausted in heart failure and unable to further respond to increasing volume while it plays an important compensatory role in adaptation to LV dysfunction in heart failure.

The fall in creatine levels and creatine kinase isozyme changes in the failing heart are reversible: complex post-transcriptional regulation of the components of the CK system.

Decreases in total creatine kinase (CK) activity and creatine [Cr] combine to limit the capacity of the failing heart to rapidly re-synthesize ATP (energy reserve). If the loss in energy reserve could be reversed, cardiac contractile reserve may be improved. Here we test whether these changes are reversible during recovery from heart failure. Left ventricular (LV) contractile function was measured in chronically instrumented conscious dogs with heart failure (CHF) induced by cardiac pacing for 3-4 weeks, and after recovery from heart failure (Recovery) (unpaced) for 5-6 weeks. LV contractile function and contractile reserve were depressed in CHF but returned to control in Recovery. CK capacity fell by 55% in CHF due to decreases in [Cr] (-39%) and CK activity (-25%), but was fully restored in Recovery. CK-B isozyme activity, protein (Western) and mRNA levels (real time PCR), respectively, were higher by 2-, 5.4- and 11-fold in CHF and higher by 3-, 2- and 2-fold in Recovery. CK-MM activity was decreased (-30%) in CHF but returned to normal levels during Recovery; CK-M protein was 30% lower in both CHF and Recovery even though there were no changes in mRNA levels. A similar pattern was found for mitochondrial CK (sMtCK). Deceases in CK activity and [Cr] in CHF are reversible. Decreases in CK-MM and sMtCK activities, but not the increases in CK-BB and CK-MB, also reversed. Neither the changes in protein nor mRNA levels for CK-B and CK-M correlated to their activities, suggesting that CK is under complex post-transcriptional regulation.

Sodium channel enhancer restores baroreflex sensitivity in conscious dogs with heart failure.

We compared the cardiac inotropic, lusitropic, and chronotropic responses to the Na(+) channel enhancer LY-368052 in conscious dogs before and after development of congestive heart failure (CHF). We also examined the effect of LY-368052 on baroreflex sensitivity and the efferent neural mechanisms of the bradycardic response in heart failure. Dogs were chronically instrumented, and heart failure was induced by right ventricular pacing at 240 beats/min for 3-4 wk. LY-368052 dose-dependently increased left ventricular contractile performance before and after the development of CHF to a similar extent. The inotropic effect of LY-368052 in heart failure was not altered by either ganglionic or beta-adrenergic receptor blockade. LY-368052 improved cardiac relaxation and induced bradycardia in dogs with heart failure but not in normal dogs. The negative chronotropic effect of LY-368052 was eliminated by ganglionic blockade but not beta-adrenergic blockade, suggesting that the bradycardia was mediated by the autonomic nervous system via enhanced parasympathetic tone. Baroreflex sensitivity was assessed as the pulse interval-mean arterial pressure slope in response to temporary pharmacological (nitroglycerin or phenylephrine) and mechanical (brief occlusion of inferior vena cava) alterations of arterial pressure in conscious dogs before and after development of heart failure. Baroreflex sensitivity was significantly depressed in heart failure and restored completely by acute treatment with LY-368052. Thus the Na(+) channel enhancer LY-368052 maintains its beta-receptor-independent inotropic effect in chronic CHF and specifically improves ventricular relaxation and depressed baroreflex function.

Combined inotropic and bradycardic effects of a sodium channel enhancer in conscious dogs with heart failure: a mechanism for improved myocardial efficiency compared with dobutamine.

We compared the cardiac inotropic, chronotropic, and myocardial O(2) consumption (MVO(2)) responses to the sodium (Na(+)) channel enhancer, LY341311 [(S)-4-[3-[[1-(diphenyl-methyl)-3-azetidinyl]oxy]-2-hydroxypropoxy]-1H-indole-2-carbonitrile monohydrate], with the beta-receptor agonist dobutamine in conscious dogs with heart failure. Heart failure was induced in chronically instrumented dogs by right ventricular pacing at 240 beats per minute for 3 to 4 weeks. LY341311 (10-100 microg/kg/min i.v.) dose dependently increased cardiac contractile function as reflected, at the highest dose, by increases in left ventricular dP/dt(max) (55 +/- 7%), and fractional shortening (62 +/- 9%), accompanied by increases in cardiac stroke work (111 +/- 18%) and minute work (34 +/- 10%) and decreases in heart rate (33 +/- 4%). Dobutamine (2-15 microg/kg/min i.v.) increased contractile responses to a similar degree but also increased heart rate (15 +/- 5%) at the highest dose. Complete ganglionic blockade with hexamethonium and atropine or with hexamethonium alone abolished the bradycardic effect but not the inotropic response to LY341311. At similar levels of inotropic response, dobutamine (10 microg/kg/min) increased MVO(2) by 23 +/- 7% (P < 0.05), whereas LY341311 (100 microg/kg/min) had no effect. In the presence of left atrial pacing at a constant heart rate and at matched contractile work, MVO(2) was increased by LY341311 to the same extent as dobutamine. These data indicate that autonomically mediated bradycardia produced by LY341311 contributes to a favorable net metabolic effect on myocardial O(2) utilization in the failing heart while providing inotropic support comparable to a beta-receptor-mediated agonist.

A sodium channel enhancer, LY341311, increases myocardial contractile performance without increasing heart rate in conscious normal dogs: a comparison with dobutamine.

BACKGROUND: Catecholamines and many inotropic agents increase cardiac contractility but also cause excessive myocardial O2 consumption (MVO2). We determined if the novel Na+ channel enhancer LY341311, which increases myocardial contractility independent of beta receptors, can produce significant cardiac inotropic effects compared with dobutamine but at lower oxygen cost in conscious dogs. METHODS AND RESULTS: Mongrel dogs were chronically instrumented for measurement of arterial pressure, left ventricular (LV) pressure and internal diameter, coronary blood flow, and arterial and coronary sinus O2 content. Both LY341311 and dobutamine produced dose-dependent increases in LV dP/dt, dP/dt/40, fractional shortening, and cardiac stroke work and minute work estimated from the LV pressure-diameter loop. The major difference between LY341311 and dobutamine was an opposing effect on heart rate with LY341311 slightly reducing it but dobutamine markedly increasing it. LY341311 caused a significantly smaller increase in MVO2 than dobutamine (P <.05) and produced similar cardiac inotropic effects, yielding a higher cardiac mechanical efficiency than dobutamine. However, after pacing to match heart rate with dobutamine LY341311 increased MVO2 markedly, approaching the same level as with dobutamine. CONCLUSIONS: The novel Na+ channel enhancer LY341311 caused significant increases in myocardial contractility and contractile performance without increasing heart rate. It had a beneficial energetic effect on the heart with significantly less O2 cost and improved cardiac mechanical efficiency.